Genomics at Agilent: Driving Value in DNA Sequencing

 

By Darlene J.S. Solomon, Ph.D., Agilent Chief Technology Officer, and
Emily M. Leproust, Ph.D., Agilent Director of Applications and Chemistry R&D - Genomics

		1. Innovating the NGS workflow 2. At the foundation: oligonucleotide synthesis 3. Future directions in multiplexed genomic analysis Download PDF (134k)
	Darlene J.S. Solomon, Ph.D.		Emily M. Leproust, Ph.D.

Agilent innovations help unleash imaginations of researchers. Projects in Agilent Laboratories, our central advanced research facility, begin as an exploration of a very high-risk technology, that if successful, would really drive the fundamental questions that researchers could ask and answer. When Agilent business units continue R&D and commercialize products based on Labs research, innovations typically represent 10, 100 and even 1,000 fold improvements in performance or ease of use over the previous solutions.

Agilent R&D teams include broad and deep technical expertise because to solve the problems that really change the game requires innovation at the interfaces across multiple science and engineering disciplines. We strive to stay enough ahead of where researchers are today so that we can develop the new technology into a robust product in the timeframe for their next-generation research challenges. Our teams collaborate extensively with thought leaders in academic, government and industrial research venues so that Agilent’s R&D investments will fuel the right products at the right time for emerging market needs. These relationships also augment and validate the value of our research.

One example of Agilent’s significant R&D focus is development of world-class oligonucleotide synthesis, which is at the foundation of target enrichment in simplifying DNA sequencing workflows. We provide oligos from the kilo scale to fractions of femtomoles – and in multiplexed mixtures from 2 to 55,000 unique sequences in oligo libraries. Whereas traditional methods can synthesize very good 40- and 60-mer oligos, only Agilent can supply researchers with highest quality oligos of length up to 200-mers, continually advancing genomics and the understanding it will bring.

Although the evolution of powerful instrumentation is revolutionizing genome sequencing, users still face fundamental challenges: full genome sequencing that is still relatively expensive; sample preparation that is costly, time-consuming and complex; and enormous amounts of data that require unprecedented levels of storage and analysis.

1. Agilent’s innovations to the sequencing workflow include targeted sequencing towards specific regions of a genome, simplifying sample preparation and streamlining data analysis to best achieve understanding. These three areas are especially important now that genome sequencing is far less expensive than it was previously but still orders of magnitude more costly than researchers desire.

2. Oligonucleotide synthesis is the foundation of Agilent’s target enrichment solutions for sequencing. These innovative technologies result from the company’s investments in ongoing research to develop solutions to problems customers will face next.

3. Agilent charts future directions in multiplexed and high throughput genomic analysis and is committed to the entire innovation process to move inventions out of our research laboratories and into products and solutions that advance our customers’ research goals. 

1. Innovating the NGS workflow

Agilent offers a portfolio of workflow products to make genomic experiments run faster, better and/or more cost effectively. We help scientists get their data quickly and move on to their next follow-up experiment. In this article, we highlight workflow contributions that Agilent is providing to help advance the rapidly evolving field of DNA sequencing, today and into the future

Targeting specific genomic regions

Life science researchers can target genomic regions of interest with the Agilent SureSelect Target Enrichment System. This platform enables researchers to increase the speed and cost-efficiency of next-gen sequencing workflows, conduct studies that were previously unfeasible due to the rarity of DNA sample, sequence a larger number of samples and increase throughput with automation. We developed the SureSelect Target Enrichment System by combining the depth of our in situ array technology with innovative bioreagents from Agilent’s Stratagene division and our state-of-the-art lab automation capabilities.

The Agilent SureSelect Target Enrichment System is based on an in-solution design that uses biotinylated RNA “baits” to fish targets out of a “pond” of DNA fragments. Customer-specified libraries are generated in solution, of up to 55,000 biotinylated RNA probes targeting up to 6.8Mb of genomic regions (~10Mb before repeat masking), delivered in single tubes. The capture probes are 120 base pairs long, the longest currently on the market for this application. This length increases the probe effectiveness at capturing DNA that contains unknown mutations, such as single nucleotide polymorphisms, insertions or deletions. Because the protocol is tube-based, researchers can readily automate and scale the platform for studies from 10 reactions through thousands, providing unmatched flexibility for researchers.

“The SureSelect Target Enrichment product we tested is very mature, and the uniformity of the capture went up when compared to the proof-of concept experiment published earlier this year," said Kelly Frazer, Ph.D, Professor and Chief, Division of Genome Information Science, Department of Pediatrics, UC San Diego School of Medicine. "In the end, this comes out to less sequencing for an increased sensitivity to detect variants. With these experiments, we now understand better the impact of probe design and tiling frequency which are key parameters to improve capture uniformity and SNP calling." In-solution Method for Streamlining Next-Generation Genome Sequencing Shown to be Uniform, Reproducible across Many Samples, November 17, 2009

Benefits of the Agilent SureSelect Target Enrichment System, which employs a method licensed from the Broad Institute of Harvard and MIT, include the following:

• An open platform that offers optimized protocols for all market leading next-generation DNA sequencing systems;
  
• Multiplexed DNA sequencing of 12 to 16 samples per lane, depending on the next-generation sequencing platform, reducing costs and increasing throughput up to 16-fold without compromising data quality;
  
• Small sample size requirement of 1 to 3 micrograms of genomic DNA, 10-fold less input DNA than competing products;
  
• Highly uniform coverage of sequence targets that is reproducible across a large number of samples;
  
• Scalability and format well-suited for automation.

Researchers using the Agilent SureSelect Target Enrichment System are discovering links between specific genes and mutations to syndromes such as cleft palate and diseases such as leukemia.(1)  

"The SureSelect All Exon Kit enrichment for the Applied Biosystems SOLiD System [by Life Technologies Corporation] worked in our hands extremely well," said Dr. Michal Schweiger, M.D., Ph.D., Cancer Genomics Group of the Max Planck Institute for Molecular Genetics. "The amount of input DNA required is very low and the protocol straightforward to perform. We are very happy about the technology, especially in regard to our applications studying cancer where we have limited amounts of input DNA." Agilent Technologies' Next-Generation-Sequencing Human Exome Target Enrichment Portfolio Now Available for SOLiD™, November 3, 2009

 

2. At the foundation: oligonucleotide synthesis

Disruptive technology contributions often enable new applications and new addressable markets. Our world-class core competency in synthesizing nucleic acids began in the early 1990s when we initiated research towards the fabrication of DNA microarrays by inkjet printing of nucleic acids. Since then we’ve invested steadily and enabled some of the most significant improvements in nucleic acid synthesis in recent decades.

Our proprietary capability to make very long, high quality DNA and RNA cost effectively has enabled us to provide new capabilities beyond microarrays. These include the Agilent SureSelect Target Enrichment System and custom manufacturing of oligos to supply the pharmaceutical and biotech industry’s needs for emerging RNA-based therapeutics. When researchers realize they can access tens of thousands of very high quality oligos at less than one cent per base, they begin to envision experiments that previously would not have been considered possible.

A DNA oligo with a specific base sequence must be synthesized serially, one nucleotide at a time, as shown in the figure below. To achieve high fidelity oligos, the synthesis needs to address deblock and coupling yields as addition of each base extends the oligo polymer. Typically, while coupling can be high and any errors compensated by capping, the deblock step has been a limitation. Because this step is reversible, it can lead to misincorporation of bases resulting in random deletions. In addition, the depurination side reaction needs to be eliminated to achieve long lengths; it has been the main limitation preventing routine synthesis of oligos more than 100 base pairs.

For example, the actual full length yield of a 100-base oligo using conventional synthesis processes is less than 10 percent; however, we can increase the full-length yield of SureSelect Target Enrichment System’s 120-mers to roughly 60 percent by addressing the depurination side reaction, increasing deblock yield from 98.0 to 99.5 percent efficiency and making coupling yields quantitative.

Online sequence design

Researchers can create custom Agilent SureSelect Target Enrichment System kits specifically for their studies on Agilent’s web-based design tool, eArray, the same capability Agilent uses for catalog kits. Whether the goal is to capture a specific set of exons on the X chromosome or a number of defined regions of interest based on genome-wide association studies, eArray contains many key genomes and allows biologists to upload their own sequences. Customized experiments can be designed without up-front design fees or the need to invest in specialized bioinformatics tools. eArray’s design algorithms have been proven to work over a diverse set of genomic locations, such as small and large exons, short and long contiguous genomic targets, genome targets within repeat areas and non-coding DNA.

Also available is the SureSelect Target Enrichment System Human All Exon Kit that targets all human exons in a single tube. It covers 1.22 percent of human genomic regions corresponding to the NCBI Consensus CDS Database (CCDS), including more than 700 human miRNAs from the Sanger v13 database and more than 300 additional human non-coding RNAs in a single tube.

"The sequencing results we are getting using the SureSelect Human All Exon Kit in collaboration with Agilent are promising; we believe we can use SureSelect to scale the Broad's exome sequencing efforts moving forward," said Stacey Gabriel, Ph.D., Co-Director, Genome Sequencing and Analysis Program and Medical and Population Genetics Program of the Broad Institute of MIT and Harvard. Agilent Technologies Adds Human Exon Kit to Next-Generation-Sequencing Target Enrichment Portfolio, September 22, 2009

Simplified Sample Preparation

Agilent provides specialty reagents and everyday essentials for purifying, quantifying, amplifying and analyzing nucleic acids. Examples for genomics sample prep include PCR enzymes to amplify a single or few copies of a piece of DNA and reverse transcriptases to transcribe single-stranded RNA into double-stranded DNA.

The Agilent 2100 Bioanalyzer is a microfluidics-based platform for sizing, quantification and quality control of DNA, RNA, proteins and cells. In sequencing, the bioanalyzer has quickly become the gold standard in DNA quality assessment, just as it has been for many years in validating RNA quality for microarray-based experiments. Miniaturization and digitizing of analytical instrumentation offer many advantages over conventional techniques, including improved data precision and reproducibility, short analysis times, minimal sample consumption, improved automation and integration of complex workflows.

Automated Sample Prep and Sample Enrichment

The Bravo Automated Liquid Handling Platform provides robotics for next-generation sequencing workflows to automate sample preparation and sample enrichment prior to sequencing. Automation enables faster, higher quality and more reproducible sequencing compared to manual preparation, which is more time-consuming and a source of variability.

Key Benefits of the Bravo Automated Liquid Handling Platform Decreases sample process time from 3 weeks to 1½ weeks Decreases sample to sample variability Increases capacity from 12 to 96 samples per technician

The Bravo nine-plate position footprint easily fits inside a standard laminar flow hood, enabling automated liquid handling for genomic applications. The unit automatically performs multi-channel pipetting and serial dilutions in 96- and 384-well formats. It also can dispense to a single well. Researchers can customize the deck easily to meet a wide range of applications including general liquid handling, serial dilutions, PCR preparation and cleanup, cell-based assays, filtration assays, magnetic bead based separations.

Analysis

After a next-generation system has sequenced the sample, researchers need to analyze and extract knowledge from the raw data. They also may need to integrate other heterogeneous data from previous experiments and draw conclusions that cross boundaries of application disciplines, such as genomics, genetics, metabolomics, proteomics and biomarker screening.

The Agilent GeneSpring Analysis Platform offers an expanding suite of integrated software applications for established and emerging technologies. GeneSpring integrates data and results from multiple applications so researchers can quickly analyze, compare and view results from different experiments in a single user interface.

The modular data storage architecture of GeneSpring handles data from applications such as genotyping, microarray-based comparative genomic hybridization, biomarker profiling, ChIP-on-chip location analysis, gene expression profiling, mass spectrometry-based proteomics and next-generation sequencing.

GeneSpring displays data in ways that help researchers conceptualize the information in their data and convey it to their peers. The various types of plots, graphs and diagrams highlight different aspects of the data and allow researchers to extract visual information in multiple ways.

Agilent has been supporting the GeneSpring Analysis Platform for 10 years, expanding our informatics capabilities to enable researchers to accelerate discovery and draw conclusions from studies that intersect scientific boundaries. After researchers analyze their results and compare these with their beginning hypothesis, they can move quickly to design more content with SureSelect and begin their next experiment.

3. Future directions in multiplexed genomic analysis

 
Agilent’s two unique platforms – microarrays and oligonucleotide libraries – provide three powerful directions for innovation:

• Complex oligo libraries are new tools that researchers already have applied to enable compelling new measurement capabilities and will apply to a wide variety of new applications yet to be invented. Last year, highly regarded scientific journals published articles from our early access customers about applications for oligo libraries, such as messenger RNA profiling, gene silencing, methylation analysis, histone analysis and mutagenesis.(2) Working with these collaborators provides a pipeline of new SureSelect Target Enrichment System applications.
  
• Solutions to enable researchers to realize the full value of their sequencing will make it easier, faster and less expensive for them to investigate and analyze biological questions. In the past it used to be one sample per lane, and now with indexing/barcoding it could be 12 or 16 samples per lane/quad. Indexing reduces the cost of sequencing, but the upstream process to prepare the samples needs to catch up too. We’re advancing automation capabilities of preparation and capture so the throughput of the library preparation and capture matches the throughput of the sequencing machine. Agilent is working on more advances in automation to integrate the whole process fully.
  
  We’re also bringing together Stratagene’s 15 years of experience in mutagenesis with oligo libraries to launch innovative products into the pharmaceutical market. And as we combine Stratagene’s experience in bioreagents with Agilent’s measurement and manufacturing expertise, we expect to provide new instruments that will be better, faster and less expensive that those of our competitors.
  
• Diagnosticsis an important innovation direction for Agilent because of the huge drive to expand the use of microarrays beyond the research area into the clinic to enable better medicine. We believe we are in a strong position to take the next step in our dialog with the U.S. Food and Drug Administration toward pursuing clearance for a cytogenetics device based on our platform. As sequencing becomes ubiquitous and we learn more about DNA sequences in humans and other species, including the world of microbes, we will leverage this new content into better microarray design. Our fast manufacturing turnaround enables us to take advantage of this new content quickly and keep researchers and clinicians at the leading edge.

Future directions in data analysis

The GeneSpring platform will continue to be Agilent’s platform of choice to support all the different multi-omics data types researchers will need to relate together. While it will become easier to acquire enormous data sets of raw genomic information, it will be even more critical to provide bioinformatics and applications to help researchers manipulate, share and make sense of all this data.

We’re continuing to extend and advance GeneSpring’s capabilities in statistical analysis, data management and visualization to keep pace with the explosion of DNA sequence information from advancements in next-generation sequencing. Future directions for GeneSpring will enable researchers to investigate and answer an ever-wider range of biological questions.

While GeneSpring provides modeling capabilities to allow researchers to generate and explore biochemical networks, we’re also implementing a pathway level of integration through open source -- adding to and integrating with Cytoscape. Pathway and network diagrams help place statistical results in a biological context. Direct navigation between biological pathways and their associated genes will provide even richer user experience and systems-level insight for future research including emerging applications such as synthetic biology.

Steps toward synthetic biology

Synthetic biology - the redesign of biological systems and their component parts for useful and practical purposes - is an important emerging field. Because it combines multiple disciplines, synthetic biology relies upon many experimental approaches, reagents and measurement solutions. It is already emerging as a new paradigm for chemical manufacturing, and many believe biofuels produced through synthetic biology will help address the energy challenges our world faces. A fundamental enabler of this field will be the ability to synthesize high quality DNA controllably with reproducibly defined sequence and length as addressed earlier. The field also will require significant technology advances for the assembly and characterization of genetic components, networks and genomes – work that leverages Agilent’s strengths in innovation.

Synthetic biology has many parallels to the electronics industry. Standardized, integrated electronic parts, devices and tools have enabled a well-developed, mature industry. Advocates of synthetic biology similarly champion development of tools and processes that will enable standardized, integrated biological parts, devices and networks to efficiently manufacture high-value chemicals, pharmaceuticals, bio-fuels and nanomaterials. Applications in alternative electronics and DNA based computation also are advancing throughout the world.

Many thought leaders in the field believe that when efficient DNA assembly methods and validation tools are developed, gene synthesis will become technologically accessible. In parallel, appropriate oversight measures will need to be defined and in place to ensure that use of such technology is in the interest of mankind. Enabling such capability would greatly benefit a broad range of research areas and significantly influence the direction of these areas well into the 21st century.

Conclusion

Agilent developed proprietary capability in oligonucleotide synthesis by pushing the boundaries with innovation at the edge of the spectrum. Every day we make hundreds of millions of unique oligos at extremely long length and relatively low cost at much higher quality than what is otherwise commercially available. This foundation in oligo synthesis is one example of what we refer to as win- win- win innovations. Customers win with access to new technology to investigate questions they couldn’t address or answer before. Science wins because Agilent solutions enable better understanding of biology and the world, and Agilent wins because we recoup our investments to continue innovating future measurement solutions that advance the leading edge.

Agilent is committed to accelerating advancements in DNA sequencing. We continue to collaborate with scientists and thought leaders to develop innovative solutions to problems of the next year and the next decade. We are charting future directions and supporting the entire innovation process to get inventions out of our laboratories and into products and solutions that create real value for our customers.

August 2010

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References:  

(1) Research With Agilent SureSelect Target Enrichment System

“Massively parallel sequencing of exons on the X chromosome identifies RBM10 as the gene that causes a syndromic form of cleft palate.” Johnston JJ, Teer JK, Cherukuri PF, Hansen NF, Loftus SK; NIH Intramural Sequencing Center, Chong K, Mullikin JC, Biesecker LG. Am J Hum Genet. 2010 May 14;86(5):743-8. Epub 2010 May 6.

“De novo mutations of SETBP1 cause Schinzel-Giedion syndrome.” Hoischen A, van Bon BW, Gilissen C, Arts P, van Lier B, Steehouwer M, de Vries P, de Reuver R, Wieskamp N, Mortier G, Devriendt K, Amorim MZ, Revencu N, Kidd A, Barbosa M, Turner A, Smith J, Oley C, Henderson A, Hayes IM, Thompson EM, Brunner HG, de Vries BB, Veltman JA. Nat Genet. 2010 Jun;42(6):483-5. Epub 2010 May 2.

“PHF6 mutations in T-cell acute lymphoblastic leukemia.” Van Vlierberghe P, Palomero T, Khiabanian H, Van der Meulen J, Castillo M, Van Roy N, De Moerloose B, Philippé J, González-García S, Toribio ML, Taghon T, Zuurbier L, Cauwelier B, Harrison CJ, Schwab C, Pisecker M, Strehl S, Langerak AW, Gecz J, Sonneveld E, Pieters R, Paietta E, Rowe JM, Wiernik PH, Benoit Y, Soulier J, Poppe B, Yao X, Cordon-Cardo C, Meijerink J, Rabadan R, Speleman F, Ferrando A. Nat Genet. 2010 Apr;42(4):338-42. Epub 2010 Mar 14.

“Enrichment of sequencing targets from the human genome by solution hybridization.” Tewhey R, Nakano M, Wang X, Pabón-Peña C, Novak B, Giuffre A, Lin E, Happe S, Roberts DN, LeProust EM, Topol EJ, Harismendy O, Frazer KA. Genome Biol. 2009;10(10):R116. Epub 2009 Oct 16.

(2) Research With Agilent Oligonucleotide Libraries

“Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process.” LeProust EM, Peck BJ, Spirin K, McCuen HB, Moore B, Namsaraev E, Caruthers MH. Nucleic Acids Res. 2010 May 1;38(8):2522-40. Epub 2010 Mar 22.

“High-resolution analysis of DNA regulatory elements by synthetic saturation mutagenesis.” Patwardhan RP, Lee C, Litvin O, Young DL, Pe'er D, Shendure J. Nat Biotechnol. 2009 Dec;27(12):1173-5. Epub.

“Multiplex padlock targeted sequencing reveals human hypermutable CpG variations.” Li JB, Gao Y, Aach J, Zhang K, Kryukov GV, Xie B, Ahlford A, Yoon JK, Rosenbaum AM, Zaranek AW, LeProust E, Sunyaev SR, Church GM. Genome Res. 2009 Sep;19(9):1606-15. Epub 2009 Jun 12.

“Genome-wide identification of human RNA editing sites by parallel DNA capturing and sequencing.” Li JB, Levanon EY, Yoon JK, Aach J, Xie B, Leproust E, Zhang K, Gao Y, Church GM. Science. 2009 May 29;324(5931):1210-3.

“Rapid creation and quantitative monitoring of high coverage shRNA libraries.” Bassik MC, Lebbink RJ, Churchman LS, Ingolia NT, Patena W, LeProust EM, Schuldiner M, Weissman JS, McManus MT. Nat Methods. 2009 Jun;6(6):443-5. Epub 2009 May 17.

“Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing.” Gnirke A, Melnikov A, Maguire J, Rogov P, LeProust EM, Brockman W, Fennell T, Giannoukos G, Fisher S, Russ C, Gabriel S, Jaffe DB, Lander ES, Nusbaum C. Nat Biotechnol. 2009 Feb;27(2):182-9. Epub 2009 Feb 1.

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